Choudhury, A. R. | bioRxiv Science

bioRxiv2024

Exploring the effect of Hydrocarbon Cross-linkers on the Structure and Binding of Stapled p53 Peptides

Short-length peptides are used as therapeutics due to their high target specificity and low toxicity, for example, peptides designed for targeting the interaction between oncogenic protein p53 and E3 ubiquitin ligase MDM2. These peptide therapeutics form a class of successful inhibitors. To design such peptide-based inhibitors, stapling is one of the methods in which amino acid side chains are stitched together to get conformationally rigid peptides, ensuring effective binding to their partners. In the current work, we use computer simulations to investigate p53 peptides stapled with hydrocarbon chains of different lengths and positions of attachment to the peptide. We subsequently analyze their binding efficiency with MDM2. The introduction of stapling agents restricts the conformational dynamics of peptides, resulting in higher persistence of helicity. The efficiency of the stapling agents has also been verified imposing these stapled peptides to adverse conditions viz. thermal and chemical denaturation. In addition, the conformational exploration of peptides has been investigated using Temperature replica exchange molecular dynamics (T-REMD) simulations. From both the unbiased and T-REMD simulations, p53 with a long hydrocarbon cross-linker shows a more conformationally rigid structure having high helicity compared to other stapled peptides. The rigidity gained due to cross-linking reduces the entropy of the peptide in the free state and thereby facilitates the complexation process. From the binding studies, we have shown that the peptide having multiple short staples has shown a larger enthalpy change during binding free energy, resulting from its orientation and interactions of residues in the binding interface. On the other hand, a peptide with a single long stapling agent shows less entropic penalty than other systems. Our studies suggest a plausible rationale for the relation between the length and the position of attachment of cross-linkers to peptides and the binding free energies between the peptides and their target partners.

biophysics↗

bioRxiv2020

Effect of Stapling on the Thermodynamics of Protein-Peptide Binding

Protein-protein interaction (PPI) is one of the key regulatory features to drive biomolecular processes and hence is targeted for designing therapeutics against diseases. Small peptides are a new and emerging class of therapeutics owing to their high specificity and low toxicity. For achieving efficient targeting of the PPI, amino acid side chains are often stapled together resulting in the rigidification of these peptides. Exploring the scope of these peptides demands a comprehensive understanding of their working principle. In this work, two stapled p53 peptides have been considered to delineate their binding mechanism with mdm2 using computational approaches. Addition of stapling protects the secondary structure of the peptides even in the case of thermal and chemical denaturation. Although the introduction of a stapling agent increases the hydrophobicity of the peptide, the enthalpic stabilization decreases. This is overcome by the lowering of the entropic penalty and the overall binding affinity improves. The mechanistic insights into the benefit of peptide stapling can be adopted for further improvement of peptide therapeutics.

biochemistry↗